luciferases and uses thereof

ABSTRACT

The present invention encompasses modified luciferases, methods for making modified luciferases, and assays utilizing modified luciferases. Modified luciferases of the invention show increased activity over wildtype luciferases and also show increased stability of signal. The present invention also encompasses multiplex assays utilizing multiple luciferases with different emission spectra.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/238,146, filed on Aug. 29, 2009, which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention concerns the field of luciferase reporters usefulin biological and biochemical assays.

BACKGROUND OF THE INVENTION

Luciferases are enzymes that catalyze reactions that emit light.Luciferases are named according to their source organisms such asbeetles (firefly) or marine organisms. Examples of bioluminescent marineanimals include: Renilla, also known as sea pansies, which belong to aclass of coelenterates known as the anthozoans. In addition to Renilla,other representative bioluminescent genera of the class Anthozoa includeCavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus.All of these organisms are bioluminescent and emit light as a result ofthe action of an enzyme (luciferase) on a substrate (luciferin) underappropriate biological conditions.

Different luciferases have different properties with regard to substratespecificity and intensity of light emission and stability of thebioluminescent signal, which is commonly measured by a luminometer.Luciferases are useful as transcriptional reporter genes and in imagingreporter gene expression in living subjects and many other applicationsin molecular biology.

Certain luciferases, such as those that utilize cypridina luciferin(vargulin) as a substrate, can be useful reporters because of theirstrong luminescent signal and the fact that they are secreted in thenative form. However cypridina luciferin (vargulin) is very difficult tosynthesize (usually involving an 18-step chemical synthesis). Thelimiting supply and the cost of the material have made the assaydifficult to commercialize.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides modified luciferases,methods of making modified luciferases, and methods of using modifiedluciferases.

In one aspect, the present invention provides an isolated polynucleotidethat encodes a modified Luciola Italica (also referred to as L. Italica)luciferase. In a further aspect, the modified L. Italica luciferaseshows increased luciferase activity when expressed in mammalian cells ascompared to a non human codon optimized mutant L. Italica luciferase.

In an embodiment and in accordance with any of the above, the presentinvention provides a modified L. Italica luciferase that shows anapproximately 1000-fold increased luciferase activity when expressed inmammalian cells as compared to a non human codon optimized mutant L.Italica luciferase.

In a further embodiment and in accordance with any of the above, thepresent invention provides a modified L. Italica luciferase that is ared-emitting luciferase with an emission maximum of approximately 617nm.

In a further embodiment and in accordance with any of the above, thepresent invention provides a modified L. Italica luciferase that ishuman codon-optimized.

In a further embodiment and in accordance with any of the above, thepresent invention provides a modified L. Italica luciferase that is agreen-emitting luciferase with an emission maximum of approximately 550nm.

In a further embodiment and in accordance with any of the above, thepresent invention provides a modified L. Italica luciferase thatincludes a secretory signal at its amino terminal end. In a stillfurther embodiment, the secretory signal is a chymotrypsinogen secretorysignal.

In one aspect, the present invention provides assays utilizing any ofthe modified luciferases discussed herein. In a further aspect, theassays are multiplexed reporter assays.

In one aspect, the present invention provides an isolated polynucleotidethat encodes a modified Renilla luciferase. In a further aspect, themodified Renilla luciferase shows increased activity and stability overa native human codon optimized Renilla luciferase.

In an exemplary embodiment the present invention provides a modifiedRenilla luciferase that is a green-emitting Renilla luciferase.

In a further embodiment and in accordance with any of the above, theinvention provides a modified Renilla luciferase that includes asecretory signal at its amino terminal end.

In one aspect, the present invention provides multiplexed luciferaseassays comprising at least two different luciferase reports, where theat least two different luciferase reporters emit at two differentwavelengths and/or utilize different substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data for relative luciferase stability for a Cypridinaassay conducted using reagents without sodium chloride (VLAR-1) and withsodium chloride (VLAR-1 with sodium chloride).

FIG. 2 shows data for time course of activity in a Cypridina assay using5 μl of sample (FIG. 2A) or 20 μl of sample (FIG. 2A).

FIG. 3A-B shows the sequence of a green Renilla luciferase plasmid (SEQID NO: 1).

FIG. 4 shows the sequence of a modified red firefly luciferase with asecretory signal (SEQ ID NO: 2).

FIG. 5 shows data from a Cypridina luciferase assay in varyingconcentrations of sodium chloride.

FIG. 6 shows data from a Renilla luciferase assay with and withoutstabilizer (NP40).

FIG. 7 shows data comparing luciferase activity of native human codonoptimized Renilla luciferase and a mutant Renilla luciferase of theinvention.

FIG. 8 shows data comparing luciferase activity of human codon optimizedand non-human codon optimized red-emitting L. Italica luciferase.

FIG. 9 shows data comparing luciferase activity of human codon optimizedand non-human codon optimized green-emitting L. Italica luciferase.

FIG. 10 shows data comparing luciferase activity of intracellularred-emitting L. Italica luciferase and secreted red-emitting L. Italicaluciferase.

FIG. 11 shows data comparing luciferase activity of secreted red L.Italica luciferase in the lysate and the supernatant from HEK293 cells.

FIG. 12 shows kinetics of luciferase activity in (A) Red Luciolaluciferase, (B) Guassia luciferase, (C) Cypridina luciferase, and (D)Green Renilla luciferase.

FIG. 13 shows emission spectra from (A) a double reporter assay withVargula and Red Italica luciferases and (B) a triple reporter assay withVargula, Green Renilla and Red Italica Luciferases.

FIG. 14 shows kinetics data of a Gaussia luciferase assay using a GAR-1reagent.

FIG. 15 shows data comparing stabilities of Gaussia luciferase assaysusing the GAR-2 reagent are in the presence of a stabilizer (FIG. 15A)and in the absence of a stabilizer (FIG. 15B).

FIG. 16 shows data related to relative luciferase activity of a fireflyluciferase assay.

FIG. 17 shows data of relative luciferase activity of a Cypridinaluciferase assay.

FIG. 18 shows data comparing luciferase activity of a modified Vargulaluciferase of the invention in the lysate and the supernatant frommammalian cells.

FIG. 19 shows kinetic data for luciferase activity in a Cypridinaluciferase assay.

FIG. 20 shows data comparing relative luciferase activity of GreenRenilla luciferase in the absence (top panel) and presence (bottom) of astabilizer.

FIG. 21 shows data from a firefly luciferase assay in the presence(square) and absence (diamonds) of a stabilizer.

FIG. 22 shows data from a dual assay of the invention utilizing fireflyand Cypridina luciferases.

FIG. 23 shows data from a dual assay of the invention utilizingCypridina (Panel A) and Renilla (Panel B) luciferases.

FIG. 24 shows emission spectra from a dual assay of the inventionutilizing Vargula and Green Renilla luciferases.

FIG. 25 shows data from a triple assay of the invention utilizingCypridina, firefly and Gaussia luciferases.

FIG. 26 shows emission spectra from a triple assay of the inventionutilizing Cypridina, Green Renilla and Red Italica luciferases.

FIG. 27 shows the sequence of a red firefly luciferase of the invention(SEQ ID NO: 5).

FIG. 28 shows emission spectra from a dual assay of the inventionutilizing Vargula and Red Italica luciferases.

FIG. 29 shows emission spectra emission spectra from a dual assay of theinvention utilizing (A) Gaussia and Red Italica luciferases and (B)Green Renilla and Red Italica luciferases.

FIG. 30 shows the sequence of a red emitting firefly human codonoptimized luciferase of the invention (SEQ ID NO: 3).

FIG. 31 shows the sequence of a human codon optimized green fireflyluciferase of the invention (SEQ ID NO: 4).

FIG. 32 shows the sequence of a human codon optimized Vargula luciferaseof the invention (SEQ ID NO: 6).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention. It will be apparent to one of skill inthe art that these additional features are also encompassed by thepresent invention.

Overview

The present invention provides modified luciferases and/or combinationsof luciferases, and methods of utilizing those luciferases in reportergene assays. In addition, the invention provides reagents that provideincreased stability and activity in assays using luciferase reporters.

The present invention provides modified (also referred to herein as“mutant” or “variant”) luciferases showing improved activity overwildtype luciferases or other modified luciferases known in the artreported to have improved properties for reporter gene assays or in vivoimaging applications. As used herein, “wildtype luciferases” refers toany luciferase that occurs in nature.

In certain aspects, the present invention provides modified luciferasesthat show brighter luminescence when expressed in mammalian cells ascompared to the luminescence seen when wildtype luciferases areexpressed in mammalian cells. The present invention also provides amethod of expressing luciferase as a very bright intracellular reporter(not secreted) by sequence modification to increase its utility as anintracellular reporter in multiplexed assays and for imagingapplications. The present invention also provides a composition forassays utilizing luciferases that lowers the cost and increases theefficiency and sensitivity of the assay by altering the reactionconditions such that high luminescence is produced using significantlyless amount of luciferin.

The present invention further provides reagents for assays utilizingmodified luciferases of the invention as well as mammalian expressionvectors expressing secreted and intracellular luciferases.

In further embodiments the present invention provides sequencemodifications (human codon optimization) to nucleotides encodingluciferases which result in an approximately 1000-fold increase inluciferase expression in transfected mammalian cells compared to thenon-human codon optimized versions of these genes.

In further embodiments, the invention provides novel secreted reportermodified luciferases that are about 5 to about 35 fold brighter thanwildtype luciferases. Such luciferases are used in accordance with thepresent invention as stand alone reporters or in multiplexed luciferaseassays in combination with one or more other luciferases. As will beappreciated, combinations of luciferases for multiplexed assays of theinvention can include both wildtype and modified luciferases.

In further aspects, the present invention provides assay compositionsfor measurement of modified luciferases of the invention as singleluciferase assay formats. In further aspects of the invention, assaycompositions are provided that enable simultaneous measurement of atleast two different reporters in cell lysates or supernatants using asingle assay solution. The luciferase activities of multiple reportersare analyzed by exploiting spectral differences in the emission maximaof the different luciferases.

Improved luciferases used in the present invention include withoutlimitation: (i) a red-emitting firefly luciferase (Red-Fluc) from theItalian firefly Luciola Italica (emission max 609 nm), includingintracellular (non-secreted) variants and secreted variants generated byfusing a chymotrypsinogen secretory signal sequence to the aminoterminal end of the luciferase; (ii) a green-emitting firefly luciferase(Green-Fluc) from the Italian firefly Luciola Italica (emission max 550nm), including intracellular (non-secreted) variants and secretedvariants generated by fusing a chymotrypsinogen secretory signalsequence to the amino terminal end of the luciferase; (iii) a CypridinaLuciferase or Vargula luciferase (VLuc) from the marine ostracod VargulaHilgendorfi, a secreted luciferase (emission max 395 nm or 462 nmdepending on the substrate used); (iv) Vargula luciferase that has beenmodified at the C-terminal end with a KDEL sequence (endoplasmicreticulum retention signal) so that it is expressedintracellularly-VLuc-KDEL; (v) a modified secreted blue-emitting(emission max 480 nm) Renilla luciferase (B-Rluc) which is brighter andmore stable than native renilla reniformis luciferase; (vi) a greenemitting secreted Renilla luciferase (emission max 535 nm) modified tobe secreted by fusing a synthetic secretory signal encoding genesequence in frame with the gene encoding the green emitting modified ofrenilla luciferase; (vii) a Gaussia luciferase (emission max 482 nm)either native secreted (Gluc) or modified to be expressedintracellularly (Gluc-KDEL).

Luciferases of the Invention

Modified luciferases of the present invention show increased signalmagnitude and stability. In certain embodiments, modified luciferases ofthe invention show at least a 1, 2, 3, 4, 5, 10, 50, 100, 250, 500, 750,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000-foldincrease in the magnitude of the signal over signals seen with wildtypeluciferases.

Modified luciferases of the invention may be intracellular (i.e., notsecreted), or they may be modified to be secreted. In furtherembodiments, modified luciferases of the invention are engineered tofurther express a secretory signal, general at the amino terminal end.In some embodiments, the secretory signal is a synthetic sequence. Inspecific embodiments, the synthetic sequence is MLLK VVFA IGCI WQA (SEQID NO: 7). In yet further embodiments, the secretory signal is anysignal that can induce secretion of the encoded protein, includingwithout limitation an interleukin-2 secretory signal and achymotrypsinogen secretory signal.

Vargula Luciferases of the Invention

In some aspects, the present invention provides a Cypridina Luciferaseor Vargula luciferase (VLuc) from the marine ostracod VargulaHilgendorfi, which is a secreted luciferase (emission max 395 nm or 462nm depending on the substrate used).

In further aspects, the present invention provides a modified Vargulaluciferase that shows increased signal and stability. In certainembodiments, the modified Vargula luciferase of the invention is humancodon optimized to increase expression in mammalian systems. In furtherembodiments, a modified Vargula luciferase of the invention includes awildtype or a native human codon optimized luciferase with the last twoamino acids have been mutated CQ to SN (S=serine, N=asparagine). Instill further embodiments, the present invention provides a mammalianvector expressing modified human codon optimized Vargula luciferaseexpressing intracellular Vargula luciferase. This sequence is the sameas the wildtype or native human codon Vargula luciferase with the lasttwo amino acids mutated CQ to SN (S=serine, N=asparagine) and with aKDEL (endoplasmic reticulum retention) sequence added after theC-terminal asparagine residue.

Firefly Luciferases of the Invention

In some aspects, the present invention provides a red-emitting fireflyluciferase (Red-Fluc) from the Italian firefly Luciola Italica (emissionmax 609 nm) and a green-emitting firefly luciferase (Green-Fluc) fromthe Italian firefly Luciola Italica (emission max 550 nm

In further embodiments, the present invention provides human codonoptimized sequences of red-emitting L. Italica luciferases. Such humancodon optimized red-emitting L. Italica luciferases show significantlyincreased activity over wildtype red-emitting L. Italica luciferases(see FIG. 8). In still further embodiments, the present inventionprovides human codon optimized sequences of red-emitting L. Italicaluciferases according to the sequence provided in FIG. 30 (SEQ ID NO:3). In still further embodiments, the present invention provides humancodon optimized sequences of red-emitting L. Italica luciferases encodedby polynucleotides with about 80%-99% sequence identity to SEQ ID NO: 3.In still further embodiments, the present invention provides luciferasesthat are encoded by polynucleotides with about 80%, 85%, 90%, 95%, 96%,97%, 98%, and 99% sequence identity to SEQ ID NO: 3.

In still further embodiments, the present invention provides secretedred-Italica luciferases. FIG. 10 shows a comparison of luciferaseactivity of a human codon optimized red-emitting L. Italica luciferasesfused to a chymotrypsinogen secretory signal to a non-secreted form ofthe human codon optimized red-emitting L. Italica luciferase. Asdiscussed above, a number of different secretory signals can be used toproduce secreted forms of modified luciferases of the invention.However, for red firefly luciferase, not all secretory signals produce asecreted luciferase. For example, popular signal sequences such as the Nterminal 16 amino acid sequence of Gaussia luciferase and theInterleukin 2 secretory sequence do not successfully produce a secretedform of red emitting firefly luciferase.

Fusing a chymotrypsinogen secretory signal to a human codon optimizedred-emitting L. Italica luciferases did successfully produce a secretedform of this luciferase. In some embodiments, the present inventionprovides a red firefly luciferase (also referred to herein as“red-emitting luciferase” and “red-emitting L. Italica luciferase”) thatis modified to include a synthetic secretory signal. In certainembodiments, the modified red firefly luciferase is encoded by thepolynucleotide has the sequence provided in FIG. 4 (SEQ ID NO: 2). Instill further embodiments, the present invention provides a luciferasesencoded by polynucleotides with about 80%-99% sequence identity to SEQID NO: 2. In still further embodiments, the present invention providesluciferases that are encoded by polynucleotides with about 80%, 85%,90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 2. Theunderlined portion of FIG. 4 is the secretory signal. FIG. 11 shows acomparison of luciferase activities in supernatants and lysates ofHEK293 cells transfected with a secreted red Italica Luciferase of theinvention.

In further embodiments, the present invention provides human codonoptimized sequences of green-emitting L. Italica luciferases. Such humancodon optimized green-emitting L. Italica luciferases show significantlyincreased activity over a previously described thermostable mutant ofgreen-emitting L. Italica luciferase (B. R. Branchini et al., AnalyticalBiochemistry, 361 (2): 253-262 (2007)—see FIG. 9). In still furtherembodiments, the present invention provides human codon optimizedsequences of green-emitting L. Italica luciferases according to thesequence provided in FIG. 31 (SEQ ID NO: 4). In still furtherembodiments, the present invention provides human codon optimizedsequences of luciferases encoded by polynucleotides with about 80%-99%sequence identity to SEQ ID NO: 4. In still further embodiments, thepresent invention provides luciferases that are encoded bypolynucleotides with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%sequence identity to SEQ ID NO: 4.

Renilla Luciferases of the Invention

In some aspects, the present invention provides a modified efficientlysecreted blue-emitting (emission max 480 nm) Renilla luciferase(B-Rluc),which more stable than the wildtype renilla reniformisluciferase, and a green emitting secreted Renilla luciferase (emissionmax 535 nm) modified to be secreted by fusing a synthetic secretorysignal encoding gene sequence in frame with the gene encoding the greenemitting modified of Renilla luciferase. Mammalian cells transfectedwith the secreted green Renilla luciferase mutant described here showapproximately 35-fold higher luciferase activity compared to mammaliancells transfected with the native (human codon optimized) Renillaluciferase (see FIG. 7). Further the secreted green Renilla luciferaseshows excellent stability of the bioluminescent signal (withoutcompromising signal intensity) when assayed using the Renilla luciferaseassay reagent described in this application (with the stabilizerincluded, see FIG. 7), thus making it an ideal reporter for Highthroughput screening applications.

In certain embodiments, the present invention provides a green-emittingRenilla luciferase plasmid sequence with the sequence pictured in FIG. 3(SEQ ID NO: 1).

Gaussia Luciferases of the Invention

In some aspects, the present invention provides a Gaussia luciferase(emission max 482 nm) that is either native secreted (Gluc) or modifiedto be expressed intracellularly (Gluc-KDEL). Such Gaussia luciferasescan be used in single, double and triple reporter assays as discussed infurther detail herein in combination with any of the other luciferasesdiscussed herein or known in the art.

Luciferase Assays of the Invention

In certain aspects, the present invention provides compositions thatimprove stability and signal for assays utilizing wildtype and/ormodified luciferases of the present invention.

In some embodiments, sodium chloride is added to improve the stabilityof luciferase assays of the invention. In such embodiments, aconcentration of sodium chloride is utilized that improves the stabilityof the bioluminescent signal without affecting intensity. In furtherembodiments, sodium chloride concentrations in the range of about 0.05 Mto about 1 M are used to improve stability of luciferase assays of theinvention. In still further embodiments, sodium chloride concentrationsof about 0.05 to about 0.5, 0.1 to about 0.4, about 0.2 to about 0.3,and about 0.05 to about 0.2M are used in luciferase assays of theinvention. In specific embodiments, sodium chloride is added to improvethe stability of assays utilizing wildtype and/or modified Vargulaluciferases.

In further embodiments, certain luciferase substrates are added toluciferase assays to improve the stability of the bioluminescent signal.In such embodiments, the substrate added as a stabilizer may be anadditional substrate that is not the substrate upon which the luciferaseitself acts. For example, in assays utilizing Cypridina luciferase,coelenterazine is added to the assay to stabilize the assay stability.Coelenterazine is an oxidizable luciferin that is easily prone tooxidation but is not a substrate for the Cypridina luciferase. As willbe appreciated, any luciferase assay described herein can be furthermodified by adding substrates for other luciferases as a stabilizer.

In some embodiments, the concentration of luciferase substrate isadjusted to improve the magnitude and/or stability of the signal. Infurther embodiments, low (under 1 μM) concentrations of substrate isused to improve luciferase signals. For example, for Cypridinaluciferase assays, about 1 to about 25 nM Vargulin are used in assays ofthe invention. In further embodiments, about 1-100, 5-90, 10-80, 15-70,20-60, 25-50, and 30-40 nM Vargulin are used in assays of the invention.In further exemplary embodiments, substrates for the luciferase assaysdescribed herein (including Cypridina, Gaussia and L. Italicaluciferases) are added in concentrations of from about 1 nM to about 250μM. In still further embodiments, substrates are added in concentrationof about 10 nM-200 μM, 50 nM-150 μM, 100 nm-100 μM, 150 nm-50 μM, 200nM-25 μM, 300 nM-10 μM, 500 nM-1 μM.

In some embodiments, Gaussia luciferases of the invention are used withoptimized reagents to produce increased activity. Kinetics of theGaussia luciferase assay using the GAR-1 reagent is shown in FIG. 14.Measurement of the luciferase activity in supernatants of cells(transfected with Gaussia luciferase) using GAR-1 reagent from Targetingsystems showed increased activity from Renilla luciferase assays fromanother vendor. The data in FIG. 14 is presented as an average oftriplicate determinations measured on a Turner TD2020 luminometer. GAR-1reagent has been described in detail in US Pat Appl Publ 2008074485,which is hereby incorporated by reference in its entirety and inparticular for all teachings related to assay reagents for the Gaussialuciferase assay.

In certain embodiments, Gaussia luciferase assays of the inventionutilize reagents stabilized with stabilizing agents. In one non-limitingexample, the stabilizing agents include NP40 (Sigma) and/orcoelenterazine. In certain embodiments, about 5 to about 200 μMcoelenterazine is used. In still further embodiments, about 10-150,20-125, 30-100, 40-75, 50-60 μM coelenterazine is used. In yet furtherembodiments, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 μM coelenterazine is used. Stability ofGaussia luciferase assays using the GAR-2 reagent are shown in FIG. 15.Using the GAR-2B version of the Gaussia luciferase assay reagent, thebioluminescent signal remains very stable (FIG. 15A) In the absence ofthe stabilizer, the signal intensity is a little higher initially butdecays faster than in the presence of the stabilizer (FIG. 15B). Notethat the data presented in FIG. 15A and B is an average of triplicatedeterminations measured on a Turner TD2020 luminometer. The GAR-2 andGAR-2B reagents are stabilized versions of the GAR-1 reagent discussedin US Pat Appl Publ 2008074485, which is hereby incorporated byreference in its entirety and in particular for all teachings related toreagents for Gaussia luciferase assays. The GAR-2 reagent includes thecomposition GAR-1 with an additional 30 μM coelenterazine. GAR-2Breagent includes the composition GAR-1 with and additional 75 μMcoelenterazine. Without being limited by theory, it is possible that thehigher (approximately 3-fold) signal intensity seen with the GAR-2Breagent is due to the higher concentration of coelenterazine. FIG. 12Bshows the stability of the Gaussia luciferase with the GAR-2 reagentincluding a stabilizer.

In certain embodiments, stability of firefly luciferase assays isimproved using FLAR-1 reagents (Targeting Systems). FIG. 16 shows theresults from experiments using the FLAR-1 reagent from TargetingSystems. In the experiments shown in FIG. 16, the FLAR-1 reagent wasadded to the supernatant cell culture media.

Dual and Triple Luciferase Assays

In some aspects, the present invention provides dual luciferase assaysbased on spectral resolution of two or more different luciferases. Aswill be appreciated, these assays can include different wildtypeluciferases, different modified luciferases, or a mixture of a wildtypeand a modified luciferase. Such assays rely on differences in theemission spectra of the reporters used. In further embodiments, reagentsare modified to allow for more efficient multiplexing. For example, whenGaussia luciferases are multiplexed with firefly luciferases, EDTA isomitted from the reaction mixture to allow efficient reporter activity.

FIG. 13A shows the emission spectra of a dual reporter assay utilizing aVargula and Red Italica luciferase of the invention. The luciferaseswere expressed in samples of transfected cells. The luciferases used inthe experiments pictured in FIG. 13A represent a modified red emittingfirefly luciferase of the invention that is human codon optimized andintracellular (non-secreted) and a Cypridina luciferase of the inventionthat is from Cypridina hilgendorfi modified to be human codon optimizedand secreted.

FIG. 13B shows the emission spectra of a triple reporter assay utilizingVargula, Green Renilla and Red Italica luciferases. These emissionspectra were in samples of transfected cell lysates. The Vargula andred-emitting firefly luciferases are those as described above for FIG.13A and the Green Renilla luciferase is an improved secreted Greenluciferase mutant as described in further detail herein.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesor value range endpoints are provided for embodiments, additionalembodiments are described by taking any 2 different values as theendpoints of a range or by taking two different range endpoints fromspecified ranges as the endpoints of an additional range. Such rangesare also within the scope of the described invention. Further,specification of a numerical range including values greater than oneincludes specific description of each integer value within that range.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

EXAMPLES Example 1

Transfection of Mammalian Cells with Modified Luciferases

HEK-293 cells were grown in DMEM/10% FBS (fetal bovine serum) andtransfected with plasmids expressing either the human codon-optimized ornon-human codon optimized forms of the red emitting and green emittingfirefly luciferases (from Luciola Italica) under control of the CMVpromoter. Transfections were performed using the Targefect F-2 reagent(Targeting Systems) using the manufacturers protocols. Forty eight hourspost transfection, the cells were lysed using the cell lysis reagent(CLR-1) from Targeting Systems, Santee. 20 μl aliquots of the celllysate were mixed with 100 μl of the FLAR-1 (firefly luciferase assayreagent from Targeting Systems).

Example 2

Cypridina Luciferase Assays with Increased Stability

Compositions were developed for achieving optimal performance ofCypridina luciferase assay reagents. These assays had improved stabilityof the bioluminescent signal without affecting the overall activity ofthe enzyme.

Vargulin is generally unstable and easily oxidized, making long termstorage of this substrate difficult. However, Vargulin stored in anacidic buffer (66 mM monobasic potassium phosphate, pH 6-6.5) and storedat −80° C. was very stable and did not lose activity even when storedfor several months. In contrast, Vargulin dissolved in a neutral tobasic phosphate buffer (e.g. 200 mM dibasic potassium phosphate (ph 8))is very unstable and begins to lose activity rapidly within a few hoursat room temperature. Cypridina luciferase activity was optimal when 200mM dibasic potassium phosphate was used as the reaction buffer insteadof 66 mM monobasic sodium phosphate. Hence 200 mM dibasic potassiumphosphate was used as the reaction buffer. Concentrations of be 3-6 nMVargulin were found to be effective, and these concentrations are muchlower than what is generally used in such assays (see for example Wu etal (2007) Biotechniques, 42(3):290-292).

The Cypridina luciferase assay showed increased stability when sodiumchloride was included in the reaction. For example, FIG. 1 shows therelative luciferase stability (RLS) between VLAR-1 (no sodium chloride)and VLAR-2 (VLAR-1+sodium chloride). Sodium chloride clearly stabilizedthe RLS. For the experiments in FIG. 1, 20 μl of sample was added with40 μl of VLAR solution for the assay followed by 20 μl of Vargulinsubstrate.

FIG. 5 shows a further titration experiment indicating that sodiumchloride concentrations of around 0.5M provide increased stability overcontrol reagents with no sodium. Further concentrations that are of usein stabilizing such assays include from about 25 mM to about 750 mMsodium chloride. For experiments in FIG. 5, 5 μl of the indicatedconcentrations of sodium chloride solutions were added to 35 μl of VLARbuffer (20 mM dibasic potassium phosphate, pH=8.0). The assay wascarried out by mixing 20 μl of sample with 40 μl of VLAR buffer (withsodium chloride) and then adding 20 μl of Cypridina luciferin.

Further stability of the bioluminescent signal as well as improvement inoverall luciferase activity was observed when coelenterazine, anotheroxidizable luciferin easily prone to oxidation (but not a substrate forCypridina luciferase) m was included in the assay composition. A 15minute pre-incubtion was found to result in increased stability of thebioluminescent signal using sample volumes between 5 and 20 μl (roughly40% drop in 26 minutes using an assay volume of 20 μl and 15% drop in 26minutes using an assay volume of 5 μl—see FIGS. 2A and 2B. Aconcentration of coelenterazine that worked well to stabilize thereagent was 15 μM . Concentrations in the range of about 10 μM to about50 μM can also be used. The inclusion of coelenterazine in thecomposition decreased the background of the assay by more than 10-fold(background reading dropped form 153.6 to 12.4) and also resulted in a15% increase in the intensity of the bioluminescent signal. Controls inwhich buffers with identical composition (i.e., inclusion ofcoelenterazine but omission of Cypridina luciferin) showed no activity.Coelenterazine is not a substrate for Cypridina luciferase and can beused to safely reduce the background and increase stability whenCypridina luciferase is assayed alone or in combination with otherluciferases (such as firefly luciferase) which do not use coelenterazineas a substrate. For the experiments shown in FIG. 2, 5 or 20 μl of thesample (media supernatant) was mixed with 40 μl of the VLAR buffer (200mM dibasic potassium phosphate, 50 mM NaCl). The firefly and Cypridinaluciferase assay reagents can be mixed into a single solution which canbe used to efficiently measure both Cypridina luciferase and fireflyluciferase activity by spectrally resolving the luciferases usingappropriate filers. However, the DTT concentration in the fireflyluciferase assay reagent can affect activity in such situations, becausethe activity of both luciferases is decreased due to interference of DTT(present in low concentration in the firefly assay reagent with theCypridina luciferase assay (there is almost a 10-fold drop in Cypridinaluciferase activity). However, since the signal intensity of theCypridina luciferase assay is very robust, the signal is stillacceptable and improvement in Cypridina luciferase activity is observedif the DTT concentration in the firefly luciferase assay reagent isdropped to 2.5 mM (a 3 fold drop in activity of Cypridina luciferase isstill observed). Single solution based dual assays in which Cypridinaluciferase is multiplexed with Green emitting Renilla luciferase workvery well without loss of activity of either Cypridina or renillaluciferase when the two solutions are mixed.

Example 3

Renilla Luciferase Assays Utilizing Modified Renilla Luciferases andStabilizing Reagents

The secreted modified green Renilla luciferase of the present inventionshowed significantly greater activity over wildtype Renillaluciferase—see FIG. 7. For the experiments pictured in FIG. 7, HEK 293cells were transfected with expression vectors expressing either nativeRenilla luciferase or the secreted Green Renilla luciferase mutant.Cells were lysed 48 hrs post transfection and assayed for luciferaseactivity.

Assays with and without stability assay reagents for green Renillaluciferase were investigated. FIG. 6 shows that assays conducted withstabilizer showed greater stability than those without. The compositionof the Renilla luciferase assay reagent (no stabilizer) was: 30 μMcoelenterazine, 0.4× PBS (Ca, Mg free), 0.027% NP40. The composition ofthe Renilla luciferase assay reagent (with stabilizer) was: 30 μMcoelenterazine, 0.4× PBS (Ca, Mg free), 0.227% NP40. Stabilizer is 2%NP40 (a non-ionic detergent).

Example 4

Kinetics of Different Luciferases

Reactions were set up to measure the kinetics of the luciferaseactivities of different luciferases in samples of transfected cells.Luciferase activities were assayed using the luciferase assay reagentssupplied with the LiveResponse assay kit. These data are shown in FIG.12A: Red Luciola (firefly), luciferase, FIG. 12B Gaussia Princepsluciferase (this is FIG. 15C), FIG. 12C: Cypridina luciferase, and FIG.12D: Green Renilla luciferase. Data represents mean of triplicatedeterminations.

Example 5

Comparison of Expression Vectors Expressing Modified Vargula Luciferases

Transfection protocols were as follows: HEK-293 cells were grown inDMEM/10% FBS (fetal bovine serum) and transfected with plasmidsexpressing wither the human codon-optimized to non-human codon optimizedforms of native VLuc, HC-VLuc, sequence 1) or modified HC-VLucs undercontrol of the CMV promoter. Transfections were performed using theTargefect F-2 regent (Targeting Systems) using the manufacturersprotocols.

The stability of the bioluminescent signal of Cypridina Luciferaseassessed using supernatants of HEK293 cells transiently transfected withthe pCMV VLuc expression vector is shown in FIG. 17.

In FIG. 19, the stability of the bioluminescent signal of CypridinaLuciferase was assessed using supernatants from HEK 293 cellstransiently transfected with the pCMV-VLuc expression vector. Sampleswere assayed using the VLAR-2 (VLAR-1 reagent from Targeting Systemswith sodium chloride) of the Cypridina luciferase assay reagent.

Human codon optimization of the gene sequence encoding the VLuc led to a5-fold improvement of luciferase expression in HEK-293 transfected withexpression vectors containing the human codon optimized versions of thevargula luciferase genes compared to the native sequences (i.e.Non-human codon optimized sequences). Addition of the KDEL sequence atthe C-terminal end results in intracellular expression of VLuc.

Example 6

Construction of Blue-Emitting (blue shifted) and Green Emitting Mutantsof Secreted Renilla Luciferase for Use as Secreted Reporters in Singleor Multiplexed Luciferase Assays

A synthetic signal peptide was deduced by rational design: MLLK VVFAIGCI VVQA (SEQ ID NO: 7). The sequence of this signal peptide was basedon rational design using signal sequences from the secretory signalsknown in the art, including those available at:http://www.unitargeting.com/Resources/Trends07.pdf

Blue-Sifted Secreted Renilla Luciferase Mutants

Secreted mutants were constructed containing signal peptide fused toamino terminal region of the human codon optimized renilla reniformisluciferase with the following additional mutations which enable i)efficient refolding after secretion to obtain an active form of theenzyme (Loma Linda paper, cysteine 124 was mutated to alanine) andadditional mutations to cause a shift in the emission max of Renillaluciferase. MLLK VVFA IGCI VVQA-HCRLuc with following mutations C124A;N53Q; V146M. Emission maxima=475 nm

Secreted BLuc Sequence 2: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations C124A; N53Q; V146M and the following eight additionalmutations A55T, S130A, K136R, A143M, M185V, M253L, S287L. The 8additional mutations increase intensity of the bioluminescent signal(Emission Maxima 475 nm)

RED SHIFTED RENILLA LUCIFERASE MUTANTS:

Secreted RLuc Sequence 1: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations C124A, D162E

Secreted RLuc Sequence 2: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations C124A; and the following eight additional mutationsAI23S/D154M/E155G/D162E/I163L/V185L F262W. Emission Maxima 535 nm

Secreted RLuc Sequence 3: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations C124A; and the following eight additional mutationsAI23S/D154M/E155G/D162E/I163L/V185L. Emission Maxima 535 nm

Example 7

Tests for Developing Assays for Vargula Luciferase

In some embodiments, different buffer solutions are used to improveassays utilizing wildtype and/or modified luciferases of the invention.In certain embodiments, a 1:1 mixture of 0.1 M Tris HCl and 75 mM sodiumphosphate is used as the assay buffer.

Several different parameters were tested to develop an assay for vargulaluciferase:

Effects of using either an acidic buffer (e.g., potassium phosphate pH5-6.8), Tris HCl pH 7.4, Tris phosphate buffer pH (8-8.5) as well asvarying assay volumes were tested. In general the use of acidicconditions significantly reduced the intensity of the bioluminescentsignal (typically 5-10 fold) while increasing the stability somewhat.Using Tris HCL ph 7.4, the activity as the assay buffer resulted in 5-10fold brighter bioluminescence but the luminescent signal was highlyunstable.

Use of a buffer mixture (1:1) of 50 mM Tris HCl, pH 7.4 and 100 mMdibasic sodium phosphate resulted in improved stability of thebioluminescent signal without compromising the intensity of thebioluminescent signal. An interesting finding was that inclusion of 0.2M NaCl further increased stability of the bioluminescent signal. Lastlythe amounts of Vargulin needed for optimal activity using this bufferedcondition are very low (1-10 nM range) making the assay extremely usefuland economical.

Increasing the concentration of Vargulin further did not increasestability of the assay further.

Stock Vargulin substrate solutions stored in an acidic condition pH(5.5-6) were relatively stable over several months when stored at −80°C.

Other parameters tested: Other stabilizers such as DTT (dithiothreitol),detergents like NP-40 or EDTA were unable to increase the intensity ofthe luminescent signal or improve stability of the assay. EDTA decreasedthe VLuc activity by at least 5-fold.

Thus one aspect of the invention concerns the following composition andvariations thereof: 20 μl of cell supernatant assays with 50 μl ofTris/phosphate buffer, pH 8, 0.2 M NaCl, 10 μl of 5-100 nM vargulin in66 mM potassium phosphate (monobasic). In certain assays, the effectiveconcentration of vargulin in the assay mix is as low as 20 nM which isapproximately 50-fold lower than that reported in the literature (seefor example Wu et al (2007) Biotechniques, 42(3):290-292)

Comparison of luciferase activity in cells transfected with vargulaluciferase with luciferase activity in cells transfected with fireflyluciferases from Photinus pyralis or Luciola Italic showed that vargulaluciferase was a much more sensitive reporter (10-20 fold improvement inbioluminescent signal compared to firefly luciferase, assay done inHEK-293 cells, all expression vectors were expressed luciferase undercontrol of the CMV promoter). An exemplary assay protocol included: 20μl aliquots of Cell supernatants (media with 5% serum) were mixed with100 μl of assay dilution buffer (50 μl of 50 mM TrisHCl, 100 mM dibasicsodium phosphate, pH 8) and 10 μl of vargulin in sodium phosphate bufferpH 6 (final concentration of vargulin in reaction mix 10-25 nM). Thesample was mixed well and bioluminescent activity was recorded in aTurner TD2020 luminometer integrated over a 20 sec time interval.

Example 8

Activity in Cell Supernatant and Cell Lysates of Cell Transfected withEither a Plasmid Vector Expressing Secreted Vargula Luciferase or anIntracellular Form of Vargula Luciferase

In cells transfected with the secreted form of modified vargulaluciferase, 80% of the activity was secreted into the cell supernatantand only 20% is cell-associated.

FIG. 18 shows intracellular and secreted Cypridina luciferase activity.Luciferase activity in cell supernatants and cell lysates of cellstransfected with a plasmid vector expressing secreted vargulaluicferase. As shown in FIG. 18 cells transfected with the secreted formof modified vargula luciferase, 80% of the activity is secreted into thecell supernatant and only 20% is cell-associated.

In cells transfected with vargula luciferase modified at the C-terminalend with a KDEL sequence, approximately 95% of the activity wasintracellular and 5% is secreted.

Example 8

Development of a Dual Reporter System Based on Blue and Red ShiftedMutants of Secreted Renilla Luciferase

Secreted mutants: Secreted mutants were constructed containing signalpeptide fused to amino terminal region of the human codon optimizedrenilla reniformis luciferase with the following additional mutationswhich enable i) efficient refolding after secretion to obtain an activeform of the enzyme (Cysteine 124 was mutated to alanine) and additionalmutations to cause a shift in the emission max of renilla luciferase:MLLK VVFA IGCI VVQA-HCRLuc with following mutations: C124A; N53Q; V146M.Emission maxima=475 nm

Secreted RLuc Sequence 2: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations. C124A; N53Q; V146M and the following eight additionalmutations A55T, S130A, K136R, A143M, M185V, M253L, S287L. The 8additional mutations increase intensity of the bioluminescent signal.Emission Maxima 475 nm

RED SHIFTED RENILLA LUCIFERASE MUTANTS: Secreted RLuc Sequence 1: MLLKVVFA IGCI VVQA-HCRLuc with following mutations: C124A, D162E

Secreted RLuc Sequence 3: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations: C124A; and the following eight additional mutationsAI23S/D154M/E155G/D162E/I163L/V185L F262W. Emission Maxima 535 nm

Secreted RLuc Sequence 4: MLLK VVFA IGCI VVQA-HCRLuc with followingmutations: C124A; and the following eight additional mutations.A123S/D154M/E155G/D162E/I163L/V185L. Emission Maxima 535 nm

A single solution dual luciferase assay based on secreted renillaluciferase blue emitting (emission max at 475 nm) and green emittingmutants (emission max at 535 nm).

The mutations in the above sequences lead to the efficient expression ofsecreted renilla luciferase in the transfected cells. The twoluciferases can therefore be used in combination as a dual reportersystem and the luciferase activity of each luciferase in the transfectedcells can be resolved by using appropriate filters. The reagentcompositions for renilla luciferase assay reagents are described Walia,US Pat Appl Publ 2008074485, entitled Enhancing a Luminescent Signal,which is incorporated herein by reference in its entirety and inparticular for all teachings related to Renilla luciferase assayreagents.

Example 9

Development of a Triple Reporter System Based On Red and Green EmittingFirefly Luciferases and Gaussia Luciferase/Renilla Luciferase

Composition of the Gaussia luciferase assay reagent (GAR-1) has beendescribed in detail in a US Pat Appl Publ 2008074485, which is herebyincorporated by reference in its entirety and in particular for allteachings related to assay reagents for the Gaussia luciferase assay. Anassay reagent useful for simultaneous measurement of all there reportersin a single solution was designed by omitting EDTA from the compositionof the Gaussia luciferase assay reagent and then including all theingredients necessary for assay of firefly luciferase in a singlecomposition. The rationale behind this is that the EDTA interferes withthe firefly luciferase assay (magnesium is an important co-factor forfirefly luciferase and EDTA chelates magnesium). The ingredientsrequired for Firefly luciferase assay included in the assay compositionwere as follows—ATP, DTT. Firefly luciferin, magnesium sulfate,magnesium bromide (helps increase brightness of luminescent signal) andphosphate buffer.

The composition of the single solution for a triple reporter assay formeasuring Gaussia luciferase or Renilla luciferase in combination withred and green emitting firefly luciferase is as follows:

0.1× PBS. 5.4 ml of 5% NP40 diluted to 1000 ml and add the following: To800 ml of the above solution add the following:

Tricine 3.227 g (20 mM) 1M Magnesium sulfate0.7H2O 2.51 ml (2.67 mMMagnesium bromide0.6 H2) (1.07 mM)-add 2.14 ml of 500 mM stock solution25 mM OTT (3.86 g) 530 μM ATP (2.72 g) CoA (0.18 g)—optional Adjust withsodium phosphate to pH 7.8 Add 940 μM D-Luciferin (fee acid)-253.81 mgCDTA-0.8289 g 940 μM D-luciferin (free acid)-253.81 mg CDTA-0.8289 g0.8M Tris (0.02 M EDTA)-43.53 ml Add GAR reagent without EDTA to a totalvolume of 1 liter Dilute 100× coelenterazine substrate with the abovesolution to 1× just before use. Use normal 3 mg/5 ml absolute alcoholacidified with 30 μl of 2N HCl)

NOTE: This assay reagent does not contain enough cell lysis reagents.Hence cells have to be first lysed using 1× Cell Lysis Buffer(compatible with use of all luciferases (prepared from 5× stock solutiondescribed below:

Dilute the 5× Cell lysis buffer described below with water to 1×concentration and add to washed cells and shake at 400 rpm for 20 minsto lyse cells.

COMPOSITION OF 5× CELL LYSIS BUFFER:

For 1 liter of Buffer 5 ml NP 40 (undiluted) 25 ml Tris HCl ph 8 1.45 gNaCl 50 ml glycerol

Example 10

Development of a Single Solution Triple Luciferase Reporter Assay Basedon Red and Green Emitting Firefly Luciferases and Vargula Luciferase

A vargula luciferase-based triple reporter system was prepared by firstpreparing the vargula luciferase assay reagent (VLAR-1) and mixing it ina 1:1 ratio with the firely luciferase assay reagent (FLAR-T) to givethe triple assay reagent TVLAR-1.

Assay protocol: To 20 μl of cell lysate add 100 μl of the TVLAR-1reagent and read in the Victor luminometer (Perkin Elmer) or Varian(Promega) using appropriate filters.

PREPARATION OF VLAR-1 REAGENT:

Composition of the Vargula Luciferase Assay reagent is described below500 ML OF 0.1 M TRIS HCL PH 8 500 ML of dibasic sodium phosphate 200 mM200 ml of 5 nM Vargulin in 66 mM potassium phosphate pH 5.5 pH of finalsolution is 8-8.5

COMPOSITION OF THE FLAR-T REAGENT

SOLUTION A: 0.1× PBS. 5.4 ml of 5% NP40 diluted to 1000 ml and add thefollowing: To 800 ml of the above solution add the following: Tricine3.227 g (20 mM) 1M Magnesium sulfate. 7H2O 2.51 ml (2.67 mM) Magnesiumbromide (0.6 H2) (1.07 mM)-add 2.14 ml of 500 mM stock solution 5 mM DTT(in some embodiments, any range between 5 mM and 30 mM can be used,including 5, 10, 15, 20, 25, 26, 27, 28, 29, and 30 mM) 530 μM ATP (2.72g) CoA (0.18 g)-optionally omitted Adjust with sodium phosphate to pH7.8 Add 940 μM D-Luciferin (free acid)-253.81 mg CDTA-0.8289 g 940 μMD-luciferin (free acid)-253.81 mg CDTA-0.8289 g 941 μM D-luciferin (freeacid)−253.81 mg CDTA-0.8289 g 0.8M Tris (0.02 M EDTA)-43.53 ml

ADD SOLUTION A to a total volume of 1 liter

NOTE: This assay reagent does not contain enough cell lysis reagents foreffective lysis. Hence cells should first be lysed, e.g., using × CellLys is Buffer (compatible with use of all luciferases (prepared from ×stock solution described below: Dilute the 5× Cell lysis bufferdescribed below with water to 1× concentration and add to washed cellsand shake at 400 rpm for 20 mins to lyse cells.

Composition of 5× cell lysis buffer:

For 1 liter of Buffer 5 ml NP 40 (undiluted) 25 ml Tris HCl ph 8 1.45 oNaCl 50 ml glycerol

Composition of Firefly luciferase assay reagent (for use of fireflyluciferase as a single reporter gene).

20 mM tricine (179.2 3.55 g) MgCo3 1.07 mM 0.55 g Magnesium sulfate 2.7mM (277 ml) 0.1 mM EDTA 20 mM DTT (4.25 g) 530 μM ATP (3 g) CoA (0.198g) Add disodium phosphate 25 g to ph 7.8 Add 793 ml water before pH 470μM D Luciferin free acid 279.2 mg 5× CCLR 307 ml

Composition of 5× CCLR: 0.8 M Tris 0.02 M EDTA pH8-156 ml Glycerol 500ml Triton X100 50 ml CDTA-7.5 m moles (2.7 g) DTT 10 mM 1.542 g totalvol 1 liter.

Example 11

Development of a Single Solution Triple Luciferase Reporter Assay Basedon Red and Green Emitting Firefly Luciferases and Vargula Luciferase

Addition of stabilizer does not significantly affect (i.e, there is verylittle decrease in signal intensity) intensity of bioluminescent signalof Renilla luciferase in supernatants and lysates. FIG. 20 (top panel)shows a Renilla assay performed with 10 μl of Renilla Lysate and 20 μlof Renilla Supernatant. Assay went as follows: 20 or 10 μl of sample(Supernatant or Lysate), 50 μl of RLAR-1 reagent (Targeting Systems).FIG. 20 (bottom panel) shows Renilla Assay was performed using the samevolumes of lysate and supernatant as in the experiments in the toppanel. Assay protocol was as follows: 10 or 20 μl of lysate orsupernatant depending on the assay, 50 μl of the RLAR-1 reagent and anadditional 8 μl of RLAR stabilizer for an increased stability profilefor a time course reading. The stabilizer lowered the initial RLUreading (decreased from approximately 9000 to approximately 7000 rlu)but showed a much higher level of stability when observed over 30minutes to 1 hour (FIG. 12C). The RLAR-1 reagent is useful for highthroughput screening (HTS) applications in which a large number ofsamples need to be assayed. In the absence of the stabilizer, the signalintensity decays faster than in the presence of stabilizer (FIG. 21).Note: Data presented is average of triplicate determinations measured ona Turner TD2020 luminometer. In FIG. 21, a time course was taken usingthe standard protocol of 10 μl lysate, 50 μl of RLAR reagent withoutstabilizer indicating drop in Renilla luciferase activity.

FIG. 22 shows the stability of the bioluminescent signal of Cypridinaluciferase and firefly luciferase using the DLAR-3 reagent. This reagentis useful for HTS applications involving both Cypridina luciferase andthe red-emitting Luciola luciferase. Note: Data presented is average oftriplicate determinations measured on a Turner TD2020 luminometer. TheDLAR-3 reagent (Targeting Systems) is a dual assay reagent based onsecreted Cypridina luciferase and a secreted or intracellularred-emitting firefly luciferase.

FIG. 28 shows emission spectra of Cypridina and Firefly luciferases insamples of transfected cells (lysates or supernatants). The emissionspectra were recorded on a Fluorolog-3 spectrofluorometer (HoribaScientific, Japan) using a liquid nitrogen cooled CCD. The luciferaseswere assayed by mixing 200 ul of the sample with the appropriateluciferase assay reagent to obtain spectral profiles. Emission max ofCypridina Luciferase is 463 nm; Red Italica 617 nm.

Example 12

Double and Triple Luciferase Reporter Assays Based on RenillaLuciferase, Firefly Luciferase and Vargula Luciferase

Kinetics of luciferase activity of different luciferase reporters usingluciferase assay reagents in the DLAR-5 system are shown in FIG. 23.Reactions were set up to measure the kinetics of the luciferaseactivities of different luciferases in samples of transfected cells.Luciferase activities were assayed using the DLAR-5 luciferase assayreagents. The decay of the renilla luciferase signal shown in Panel Babove can be greatly minimized (ie the bioluminescent signal can berendered much more stable by addition of a Renilla luciferase stabilizerto the DLAR-5 buffer.

FIG. 24 shows Emission spectra of different luciferases in samples oftransfected cell lysates. Relative luciferase activities of Cypridina,Green Renilla luciferases were assayed with the appropriate luciferaseassay reagent to obtain spectral profiles. The emission max of Vargulaluciferase is 463 nm; Green Renilla luciferase is 527 nm. Note that thedata presented in this application is performed with the green-emittingmutant that emits at 527 to 530 nm (this is the variation in emissionmaxima seen and the luciferase is different in sequence, properties andemission maximum from the 535 nm emitting intracellular green emittingRenilla luciferase mutant described in US Patent Publication No.20090136998, which is hereby incorporated by reference in its entiretyand in particular for all teachings related to Green Renilla luciferase.

FIG. 25 shows kinetics of luciferase activity of different luciferasereporters using luciferase assay reagents in the triple reporter system.Reactions were set up to measure the kinetics of the luciferaseactivities of different luciferases in samples of transfected cells.Luciferase activities were measured using the TLAR luciferase assayreagents (Targeting Systems).

FIG. 26 shows emission spectra of different luciferases in samples oftransfected cell lysates. Relative luciferase activities of Cypridina,Renilla and Red Luciola Italia luciferases were assayed with theappropriate luciferase assay reagent to obtain spectral profiles. Theemission max of Vargula luciferase is 463 nm; Green Renilla luciferaseis 527 nm and Red Luciola Italia luciferase is 617 nm.

The present invention also provides a single solution-based tripleluciferase reporter assay involving Cypridina luciferase multiplexedwith Green-emitting Renilla luciferase and Red-emitting Fireflyluciferase. This assay is compatible with high throughput applications.This assay is also optionally in a format where the three luciferasescan be assayed separately using three different assay reagents

1. An isolated polynucleotide that encodes a modified L. Italicaluciferase, wherein said modified L. Italica luciferase shows increasedluciferase activity when expressed in mammalian cells as compared to anon human codon optimized mutant L. Italica luciferase.
 2. The isolatedpolynucleotide of claim 1, wherein said modified L. Italica luciferaseshows approximately 1000-fold increased luciferase activity whenexpressed in mammalian cells as compared to a non human codon optimizedmutant L. Italica luciferase.
 3. The isolated polynucleotide of claim 1,wherein said modified L. Italica luciferase is a red-emitting luciferasewith an emission maximum of approximately 617 nm.
 4. The isolatedpolynucleotide of claim 1, wherein said modified L. Italica luciferaseis human codon-optimized.
 5. The isolated polynucleotide of claim 1,wherein said modified L. Italica luciferase is a green-emittingluciferase with an emission maximum of approximately 550 nm.
 6. Theisolated polynucleotide of claim 1, wherein said L. Italica luciferasecomprises a secretory signal at its amino terminal end.
 7. The isolatedpolynucleotide of claim 6, wherein said secretory signal is achymotrypsinogen secretory signal.
 8. An assay utilizing the modified L.Italica luciferase of claim
 1. 9. The assay of claim 8, wherein saidassay is a multiplexed reporter assay.
 10. An isolated polynucleotidethat encodes a modified Renilla luciferase, wherein said modifiedRenilla luciferase shows increased activity and stability over a nativehuman codon optimized Renilla luciferase.
 11. The isolatedpolynucleotide of claim 10, wherein said modified Renilla luciferase isa green-emitting Renilla luciferase.
 12. The isolated polynucleotide ofclaim 10, wherein said modified Renilla luciferase comprises a secretorysignal at its amino terminal end.
 13. A multiplexed luciferase assaycomprising at least two different luciferase reports, wherein said atleast two different luciferase reporters emit at two differentwavelengths and/or utilize different substrates.